Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry

Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T₄), 3,3′,5-triiodo-l-thyronine (T₃), 3,3′,5′-triiodo-l-thyronine (rT₃), 3,5-diiodo-l-thyronine (3,5-T₂), 3,3′-diiodo-l-thyronine (3,3′-T₂), and 3-iodo-l-thyronine (3-T₁) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T₁; 0.33 ng/mg TG for 3,3′- and 3,5-T₂; and 0.52 ng/mg TG for rT₃, T₃, and T₄. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT₃, T₃, and T₄. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection

Nicotine can be determined in pharmaceuticals and biological fluids by micellar liquid chromatography (MLC) using a C18 column, a mobile phase containing sodium dodecyl sulphate 0.15 M–6% (v/v) pentanol–NaH₂PO₄ 0.01 M (pH 6)–KCl 0.001 M, with electrochemical detection at 0.8 V. In the optimization step, the influence of the modifiers propanol, butanol and pentanol, and the voltage has been studied. With the proposed method the analysis time is below than 8 min, linearity better than 0.999, limits of detection and quantification (ng/ml) was 4 and 12 respectively, repeatability and intermediate precision below 1.8%, and all these parameters are adequate for the quantification of nicotine in chewing gum, dermal patches, tobacco and serum samples either by a pharmacologist, pathologist or toxicologist.     

A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA–ELISA were optimized. Under the optimal conditions, the limit of detection (IC₁₀) was 0.0067 ng/ml and the median inhibitory concentration (IC₅₀) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were ?5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring.     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles–chemiluminescence enzyme immunoassay (MPs–CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol–H₂O₂ chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs–CLEIA method had a linear range from 0.31 to 100 ng/ml with a detection limit of 0.24 ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.     

Genetic variants of estrogen beta and leptin receptors may cause gynecomastia in adolescent

Objective

Gynecomastia is a benign breast enlargement in males that affects approximately one-third of adolescents. The exact mechanism is not fully understood; however, it has been proposed that estrogen receptors and aromatase enzyme activity may play important roles in the pathogenesis of gynecomastia. While many studies have reported that aromatase enzyme (CYP19) gene polymorphism is associated with gynecomastia, only one study has shown a relationship between estrogen receptor (ER) alpha and beta gene polymorphism and gynecomastia. Thus, the aim of this study was to evaluate the relationships between CYP19 (rs2414096), ER alpha (rs2234693), ER beta (rs4986938), leptin (rs7799039), and leptin receptor (rs1137101) gene polymorphisms and gynecomastia.

Methods

This study included 107 male adolescents with gynecomastia and 97 controls. Total serum testosterone (T) and estradiol (E2) levels were measured, and DNA was extracted from whole blood using the PCR–RFLP technique. The polymorphic distributions of CYP19, ER alpha, ER beta, leptin and leptin receptor genes were compared.

Results

The median E2 level was 12.41 (5.00–65.40) pg/ml in the control group and 16.86 (2.58–78.47) pg/ml in the study group (p < 0.001). The median T level was 2.19 (0.04–7.04) ng/ml in the control group and 1.46 (0.13–12.02) ng/ml in the study group (p = 0.714). There was a significant relationship between gynecomastia and leptin receptor rs1137101 (p = 0.002) and ER beta receptor rs4986938 gene polymorphisms (p = 0.002).

Conclusions

According to our results, increased E2 level and ER beta gene rs4986938 polymorphism might explain why some adolescents have gynecomastia. Leptin receptor gene rs1137101 polymorphism might affect susceptibility to gynecomastia.     

An updated meta-analysis of endothelial nitric oxide synthase gene: Three well-characterized polymorphisms with ischemic stroke

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may influence the risk of ischemic stroke (IS), but the results are still debatable. A meta-analysis was performed to investigate the association between the eNOS gene polymorphisms in IS risk. Case–control studies on the association between the G894T, T-786C, and 4b/a polymorphisms and IS were searched up to July 2012, and the genotype frequencies in the control group were found to be consistent with the Hardy–Weinberg equilibrium (HWE). The effect summary odds ratio (OR) and 95% confidence intervals (CIs) were obtained. Meta-regression was used to explore the potential sources of heterogeneity. Funnel plots and Egger's test was used to estimate small study biases, and heterogeneity was assessed by chi-square-based Q-test and I² test. There were total 6537/6475 cases/controls for G894T, 3459/3951 cases/controls for 4b/a, and 2125/2673 cases/controls for T-786C polymorphism. For G894T and 4b/a, a significant association of 894 T allele and 4a allele with increased risk of IS was found in Asians (TT + GT vs. GG: p < 0.00001, OR = 1.60, 95% CI = 1.38–1.79, P_{heterogeneity} = 0.11; aa + ba vs. bb: P < 0.00001, OR = 1.60, 95% CI = 1.30–1.97, P_{heterogeneity} = 0.02), but not in Caucasians (TT + GT vs. GG: P = 0.60, OR = 0.94, 95% CI = 0.75–1.19, P_{heterogeneity} = 0.002; aa + ba vs. bb: P = 0.13, OR = 0.81, 95% CI = 0.62–1.06, P_{heterogeneity} = 0.63). For T-786C polymorphism, there were no significant differences in genotype distribution between IS and control in Asians (CC + TC vs. TT: P = 0.15, OR = 1.14, 95% CI = 0.95–1.37, P_{heterogeneity} = 0.94) and in Caucasians (CC + TC vs. TT: P = 0.72, OR = 0.96, 95% CI = 0.75–1.22, P_{heterogeneity} = 0.53). This analysis provides strong evidence that the eNOS T-786C gene polymorphism is not associated with IS, the G894T and 4b/a polymorphisms might be associated with IS, at least in Asians.     

Hsa-miR-499 polymorphism (rs3746444) and cancer risk: A meta-analysis of 17 case–control studies

Introduction

MicroRNAs (miRNAs) are a family of endogenous, small and noncoding RNAs that negatively regulate gene expression by suppressing translation or degrading mRNAs. Recently, many studies investigated the association between hsa-miR-499 rs3746444 polymorphism and cancer risk, which showed inconclusive results.

Methodology/main results

We conducted a meta-analysis of 17 studies that included 7842 cancer cases and 8989 case-free controls and assessed the strength of the association, using odds ratios (ORs) with 95% confidence intervals (CIs). Overall, hsa-miR-499 rs3746444 polymorphism was associated with higher cancer risk in heterozygote model (AG vs AA, OR = 1.15, 95%CI = 1.01–1.30, P_{heterogeneity} < 0.001), dominant genetic model (GG/AG vs AA, OR = 1.18, 95% CI = 1.04–1.33, P_{heterogeneity} < 0.001) and allele contrast (G vs A, OR = 1.09, 95% CI = 1.01–1.18, P_{heterogeneity} = 0.021). In the stratified analyses, we observed that the GG/AG genotype might modulate breast cancer risk (OR = 1.13, 95% CI = 1.01–1.26, P_{heterogeneity} = 0.111) comparing with the AA genotype. Moreover, a significantly increased risk was found among Asian populations in heterozygote model (AG vs AA, OR = 1.23, 95% CI = 1.06–1.43, P_{heterogeneity} < 0.001), homozygote model (GG vs AA, OR = 1.22, 95% CI = 1.02–1.46, P_{heterogeneity} = 0.319), dominant model (GG/AG vs AA, OR = 1.25, 95% CI = 1.06–1.39, P_{heterogeneity} < 0.001) and allele contrast (G vs A, OR = 1.14, 95% CI = 1.04–1.25, P_{heterogeneity} = 0.021).

Conclusions

These findings supported that hsa-miR-499 rs3746444 polymorphism contributes to the susceptibility of cancers.     

Apamin inhibits hepatic fibrosis through suppression of transforming growth factor β1-induced hepatocyte epithelial–mesenchymal transition

Apamin is an integral part of bee venom, as a peptide component. It has long been known as a highly selective block Ca²⁺-activated K⁺ (SK) channels. However, the cellular mechanism and anti-fibrotic effect of apamin in TGF-β1-induced hepatocytes have not been explored. In the present study, we investigated the anti-fibrosis or anti-EMT mechanism by examining the effect of apamin on TGF-β1-induced hepatocytes. AML12 cells were seeded at ∼60% confluence in complete growth medium. Twenty-four hours later, the cells were changed to serum free medium containing the indicated concentrations of apamin. After 30 min, the cells were treated with 2 ng/ml of TGF-β1 and co-cultured for 48 h. Also, we investigated the effects of apamin on the CCl₄-induced liver fibrosis animal model. Treatment of AML12 cells with 2 ng/ml of TGF-β1 resulted in loss of E-cadherin protein at the cell–cell junctions and concomitant increased expression of vimentin. In addition, phosphorylation levels of ERK1/2, Akt, Smad2/3 and Smad4 were increased by TGF-β1 stimulation. However, cells treated concurrently with TGF-β1 and apamin retained high levels of localized expression of E-cadherin and showed no increase in vimentin. Specifically, treatment with 2 μg/ml of apamin almost completely blocked the phosphorylation of ERK1/2, Akt, Smad2/3 and Smad4 in AML12 cells. In addition, apamin exhibited prevention of pathological changes in the CCl₄-injected animal models. These results demonstrate the potential of apamin for the prevention of EMT progression induced by TGF-β1 in vitro and CCl₄-injected in vivo.     

Amelioratory effects of testosterone treatment on cognitive performance deficits induced by soluble Aβ1–42 oligomers injected into the hippocampus

This study was undertaken to investigate the protective effects and potential mechanism of testosterone (T) on cognitive performance in adult male rats given bilateral intrahippocampal injections of beta amyloid 1–42 oligomers (Aβ1–42) combined with gonadectomy (Aβ + GDX). A series of experiments were designed to verify the optimal administration time and dose of T and to explore its potential protective mechanisms on spatial ability in Aβ + GDX rats in the Morris water maze test. Aβ1–42 was injected only once two weeks before testing, while T and the androgen receptor (AR) antagonist flutamide (F) were administered daily beginning 2 days before and throughout the 6 days of testing. The Aβ1–42 injection and GDX individually impaired cognitive performance, and the combination of these treatments was additive, leading to even greater impairment. The serum T level peaked at 48 h after administration. T doses ranging from 0.25 to 1.00 mg corresponding to serum T levels of 4.5–21.35 ng/ml improved the spatial ability. Animals administered 0.75 mg of T corresponding to the serum T level of 15.2 ng/ml had the most significantly improved behavioral performances. However, higher T doses of 1.50 and 2.00 mg resulting in serum T levels of 34.8 and 45 ng/ml, respectively, impaired the behavioral performances. F had no effect on the serum T level and spatial ability, but it blocked the activational effect of T. These findings indicate that the effect of T on behavioral performances is partly mediated through ARs.     

Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in human serum by liquid chromatography–tandem mass spectrometry

The current paper describes a validated method for the detection and quantification of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018), an ingredient of a herbal mixture called “Spice”, by means of HPLC–ESI–MS–MS in serum. Lower limit of detection and lower limit of quantification were 0.07 and 0.21 ng/ml, respectively. In 2 subjects who consumed ca. 50 μg/kg of JWH-018 by smoking, the active ingredient was detected by means of the described method. Thereby, the serum concentrations reached values of approx. 10 ng/ml and dropped within 3 h very fast (<10% of the measured maximum concentrations).     

An organic–inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic–inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core–shell Fe₃O₄-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H₂O₂ as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001–200 ng mL⁻¹ AFP with a low detection limit of 0.5 pg mL⁻¹ at 3s_B. Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL⁻¹ AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay.     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

Corpus luteum function following single and double ovulation during estrous cycle in Sanjabi ewes

This study compared the effect of double and single ovulation on serum progesterone concentrations and luteal characteristics in Sanjabi ewes at different days of the estrous cycle. The estrous cycles of 197 Sanjabi ewes were synchronized by a 12-day treatment with intravaginal sponges (Chronogest^®). Estrus was detected in 144 ewes 27–39 h after sponge removal. Daily blood samples were taken every morning and analyzed for serum progesterone (P4). Ewes were then transported to a local abattoir, where nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovarian follicles were measured and categorized by size (very small <2 mm; small 2–3.5 mm; medium 3.5–5 mm; large >5 mm). On each slaughter day, the number of corpora lutea per ewe was classified as single and double ovulation. The results show that the effect of dominant follicles was less during the mid-luteal phase. Ovulation rate of right, left and both ovaries were (54.9%), (23.6%) and (21.5%), respectively. The incidence of double ovulations was 40.2%. In the case of ewes exhibiting double ovulation, 46.6% occurred unilateral (ewes exhibited both ovulations on the right ovary); whereas 53.4% occurred bilateral (ewes exhibited ovulations on the right and left ovaries). Unilateral double ovulation was not observed in the left ovary. The right ovary appeared to play a significantly greater role in ewes showing single and double ovulations than the left ovary (P < 0.05). Serum progesterone concentration showed minimum and maximum levels of 0.29 ± 0.15 and 5.51 ± 0.75 ng/ml on days 16 and 11 post-estrous, respectively (P < 0.001). The mean volume of individual corpus lutea in ewes with single ovulations was significantly higher than in ewes with double ovulations (P < 0.01). However, the total volume of corpus lutea in ewes with single ovulation was significantly lower than in ewes with double ovulations in some days of estrous cycle (P < 0.01). The serum progesterone concentration was significantly higher in double than single ovulating animals on days 1–16 of the estrous cycle (P < 0.001). These results indicated a relatively high incidence of double ovulation in ewes associated with increasing total luteal volume and high circulating concentrations of progesterone.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Assessment of clinical outcomes in breast cancer patients treated with taxanes: multi-analytical approach

Polymorphisms in genes encoding CYPs (Phase I) and ABCB1 (Phase III) enzymes may attribute to variability of efficacy of taxanes. The present study aims to find the influence of CYP and ABCB1 gene polymorphisms on taxanes based clinical outcomes. 132 breast cancer patients treated with taxanes based chemotherapy were genotyped for CYP3A4*1B, CYP3A5*3, CYP1B1*3, CYP2C8*3, ABCB1 1236C>T, 2677G>T/A and 3435C>T polymorphisms using PCR-RFLP. Associations of genetic variants with clinical outcomes in terms of response in 58 patients receiving neo-adjuvant chemotherapy (NACT), and chemo-toxicity in 132 patients were studied. Multifactor dimensionality reduction (MDR) analysis was performed to evaluate higher order gene–gene interactions with clinical outcomes. Pathological response to taxane based NACT was associated with GA genotype as well as A allele of CYP3A5*3 polymorphism (P_corr = 0.0465, P_corr = 0.0465). Similarly, association was found in dominant model of CYP3A5*3 polymorphism with responders (P_corr = 0.0465). Haplotype analysis further revealed A_CYP3A4–A_CYP3A5 haplotype to be significantly associated with responders (P_corr = 0.048). In assessing toxicity, significant association of variant (TT) genotype and T allele of ABCB1 2677G>T/A polymorphism, was found with ‘grade 1 or no leucopenia’ (P_corr = 0.0465, P_corr = 0.048). On evaluating higher order gene–gene interaction models by MDR analysis, CYP3A5*3; ABCB11236C>T and ABCB1 2677G>T/A; ABCB1 3435C>T and CYP1B1*3 showed significant association with treatment response, grade 2–4 anemia and dose delay/reduction due to neutropenia (P = 0.024, P = 0.004, P = 0.026), respectively. Multi-analytical approaches may provide a better assessment of pharmacogenetic based treatment outcomes in breast cancer patients treated with taxanes.